A method for achieving regeneration of soybean and its relatives from tissue culture has long been sought. Unlike such easily regenerable species as tobacco and petunia, soybean has been resistant to many prior attempts to regenerate whole plants from tissue culture. Tissue cultures are very desirable in allowing the induction of desirable traits into soybean or species capable of breeding therewith (such as G. soja) via somaclonal variation. They would also be of benefit to genetic engineers in allowing transformtion of cells by infection with Agrobacteria or by other means resulting in transformed cells in culture containing foreign DNA which could then be regenerated into whole plants bearing seed and expressing foreign genes.
D. A. Evans, et al. (eds.) (1983) in Handbook of Plant Cell Culture, vol. 1, at pp. 178-179, discuss the three possible routes available for in vitro propagule multiplication of plants in general: (a) enhanced release of axillary buds; (b) production of adventitious shoots through organogenesis; and (c) somatic embryogenesis.
Axillary bud proliferation from meristem, shoot tip, or bud cultures as a means of regeneration involves the use of an incipient shoot that has already been differentiated in vivo. Thus, to establish a complete plant, only elongation and root differentiation are required. In vitro organogenesis and embryogenesis, on the other hand, involve developmental changes: usually the formation of callus with subsequent reorganization into plantlets. This has not been easy to achieve in most plants. Evans, et al. supra at p. 178 discuss the failure of prior organogenic methods in soybean, stating that "induction of axillary bud proliferation seems to be applicable in many cases; e.g., carnation and soybean, where methods of organogenesis and embryogenesis fail."
They go on, at pp. 178-79, to state: "Although the rate of plantlet multiplication by means of organogenesis and embryogenesis is astonishing, their regeneration capacity usually diminishes rapidly after a number of subcultures, and eventually this morphogenic potential is completely lost. The initial multiplication rate for axillary bud proliferation, on the other hand, is rather slow. The rate, nevertheless, increases during the first few subcultures and eventually reaches a steady plateau during subsequent subculture cycles." These authors thus recommend axillary bud proliferation as opposed to organogenesis and embryogenesis for commercial production.
Such a bud proliferation method is described by M. S. Wright, et al., (1986) in "Plant Regeneration by Organogenesis in Glycine max", Plant Cell Reports, 5: 150-154. This method involves the germination of seeds of Glycine max (L.) on MS medium (Murashige, T. and Skoog, F. (1962) Physiol. Plant. 15: 473) containing half the recommended concentration of inorganic salts and 5 .mu.M BA (benzyladenine) also known as BAP (benzylaminopurine), CAS Registry No. 1214-39-7. Cotyledonary nodes were excised from the germinated seedlings, and non-nodal tissue removed. The piece of nodal tissue was cultured on the germination medium, then transferred to the same medium altered to contain only one fourth the recommended concentration of organic salts and 5 .mu.M BA. The nodes were subsequently subdivided and transferred to further media, and finally to soil-containing media for whole plant maturation. This method appears to be a meristemic propagation method, not going through a stage of de-differentiated cells. The article states that specific superficial regions of the cotyledonary node of soybean can be induced to become meristematic and initiate shoots, and that the constant presence of BA during culture maintains shoot morphogenesis from proregenerative tissue.
Few methods for regenerating Glycine subgenus soja, comprising G. max (soybean) and G. soja from tissue culture have been developed, although greater success has been achieved with wild relatives such as G. canescens and G. clandestina.
D. F. Hildebrand, et al., (1986), in a review article, "Soybean [Glycine max (L.) Merr.]," Biotechnology in Agriculture and Forestry Vol. 2: Crops I (Y. P. S. Bajaj, ed.) 283-308, (Table 4) summarizes recent in vitro regeneration work on Glycine, and at 293 cites the references discussed below under the heading "Meristem Culture."
K. K. Kartha, et al. (1981) "Plant Regeneration from meristems of grain legumes: soybean, cowpea, peanut, chickpea and bean," Can. J. Bot. 59: 1671-1679, describe plant regeneration from shoot apical meristems of soybean on a medium containing 1 .mu.M NAA and 0.05-0.1 .mu.MBA. Whole plants were regenerated. Under higher concentration for BA, callus was formed but whole plant regeneration was not achieved.
T. Kameya, et al. (1981), "Plant Regeneration from Hypocotyl Sections of Glycine Species," Plant Sci. Lett. 21: 289-294, disclose the use of hypocotyl sections from seedling G. canescens and G. tomentella, cultured on MS medium supplemented with NAA and BA at various concentrations to regenerate normal plants. From the eight species tested including G. max and G. soja, regeneration of shoots at high frequency was observed only from hypocotyl sections of G. canescens using 1-5 mg/l (5-25 .mu.M) BA.
T. Y. Cheng, et al. (1980), "Plant Regeneration from Soybean Cotyledonary Node Segments in Culture," Plant Sci. Lett. 19: 91-99, report the stimulation of multiple shoot-bud formation of soybeans in culture using conditioned cotyledonary node segments from seedlings. The medium used contained 0.25 .mu.M of the auxin IBA (indole butyric acid) and 5-50 .mu.M BAP. This method did not involve the formation of callus, but rather the use of explants. Concentrations of BAP higher than 10 .mu.M inhibited the development of main shoots and roots, and shoot buds formed at the cotyledonary node region. It is not clearly reported that whole plants capable of independent growth in soil were regenerated.
H. Saka, et al. (1980), "Stimulation of Multiple Shoot Formation of Soybean Stem Nodes in Culture," Plant Sci. Lett. 19: 193-201, similarly describe the formation of shoot-buds on stem nodes or apices of G. max using a culture medium containing the auxin IBA and 5-50 .mu.M BAP. Callus formation was reported which interfered with shoot bud formation. Neither emergence of new meristems from callus tissue nor whole plant regeneration were reported.
None of the foregoing references describe an organogenic regeneration method in which a tissue culture capable of producing new meristemic centers can be maintained.
In addition to the foregoing references cited in the Hildebrand, et al. review article, the following are illustrative of the state of the art.
J. M. Widholm, et al. (1983), "Shoot Regeneration from Glycine canescens Tissue Cultures," Plant Cell Reports 2: 19-20, report shoot induction from calli obtained from cotyledons and hypocotyls of G. canescens using several media including media containing NAA and 5 mg/l (25 .mu.M) BAP. Whole plants were not regenerated, and root formation was infrequent.
W. D. Beversdorf, et al., in "Degrees of Differentiation Obtained in Tissue Cultures of Glycine Species," (1977) Crop Sci. 17: 307-311, reported obtaining compact nodules of meristem-like cells which they called "growth centers." Using an induction medium containing 2,4-D (2,4-dichlorophenoxyacetic acid) and/or NAA (alpha-napthaleneacetic acid) with 0.5 mg/l kinetin (6-furylaminopurine) to culture hypocotyl sections of G. max and G. soja, Beversdorf, et al. achieved "growth centers," but no further development into plantlets.
C. A. Newell, et al. (1985) "Protoplast culture and plant regeneration in Glycine canescens," Plant Cell Tissue Organ Culture 4: 145-149 describe the regeneration of whole plants of G. canescens from protoplasts taken from seedling hypocotyl tissue. The shoot-inducing medium contained BA at 0.4 mg/l (2 .mu.M) and NAA at 0.1 and 1.0 mg/l in some experiments reported.
None of the foregoing describe the culturing of immature embryos including G. max embryos in a medium containing high BAP or other cytokinin to obtain organogenic regeneration of whole plants.
Recent works by the inventors hereof are:
Master's Thesis by Usha B. Barwale, "Screening of Soybean Cultivars for Plant Regeneration Potential and Regeneration of Soybean Plants from Undifferentiated Tissue," cataloged by the University of Illinois Library Mar. 16, 1986. This thesis defines the organogenic medium used in this invention and the development of plants therefrom.
U. B. Barwale, et al. (1986) in "Screening of Glycine max and Glycine soja Genotypes for Multiple Shoot Formation at the Cotyledonary Node," Theor. Appl. Genet. 72: 423-428, described the germination of seeds of 178 genotypes in a B5 medium comprising 1 or 5 .mu.m BAP, and counted the number of shoots formed at the cotyledonary node.
H. R. Kerns, et al. (1986), "Correlation of cotyledonary node shoot proliferation and somatic embryoid development in suspension cultures of soybean (Glycine max L. Merr.)", Plant Cell Reports 5: 140-143 disclose the induction of embryos on tissue derived from hypocotyl and cotyledon tissues from germinated seeds using a suspension medium not containing a cytokinin. Embryo formation appeared to correspond with the number of shoots formed at the cotyledonary node in the previous study. No regeneration of the embryos into whole plants was reported.
U. B. Barwale, et al. (1986), "Plant regeneration from callus cultures of several soybean genotypes via embryogenesis and organogenesis," Planta 167: 473-481, report much of the work upon which this patent application is based.
A recent commonly-assigned patent application relating to a different method of Glycine regeneration was filed Aug. 4, 1986, as U.S. patent application No. 893,256 of Glenn B. Collins, et al. This application describes a method for regeneration of G. max and other Glycine species via somatic embryogenesis involving the culturing of cotyledon tissue excised from immature embryos. That application does not disclose or claim the culturing of whole embryos in a medium containing high cytokinin to obtain organogenic regeneration.